Science topic

Cytokines - Science topic

Cytokines are small cell-signaling protein molecules that are secreted by numerous cells and are a category of signaling molecules used extensively in intercellular communication.
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I'm trying to use LPS to induce inflammation in HEK293 cells.I've tried different LPS concentration and incubation time, but qPCR result showed no difference in cytokine expression. Does anyone have relevant experience?
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If I am not mistaken, HEK293 cells do not express the Toll-like receptors necessary for LPS detection.
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It's more by curiosity, but I didn't find a convincing mechanism for explaining the opposite effects of IL10 (inhibiting [1]) versus IL6/IL23/IL21 (potentiating) Th17 differentiation on T cells. All these cytokines signal (mainly) through STAT3 which, once phosphorylated, is known to activate Th17 associated transcription factors, so one should expect that IL10 also increases Th17 differentiation, but it's not the case...
Several hypotheses could be:
- that the receptors for these cytokines differentially activate parallel signaling pathways on T cells (such as p38 Map kinase, other STATs). However, several papers claim that the anti-inflammatory effect of IL10 is through STAT3, but they only quote effects on macrophages, not on T cells ... [2]
- that the receptors phosphorylate different loci in STAT3 (and indeed there are different ones [3]), but I didn't find literature on their role on Th17 differentiation
- that IL10R/signaling is not inhibited by SOCS3 while IL6/23/21 is. One could say then that IL6/23/21 induce SOCS3 that suppresses IL6/23/21 signaling but not IL10 signaling, and that IL10 could further increase the amount of SOCS3 in Th17 for instance. But then, would low IL10 doses promote Th17 differentiation ?
Would you have some papers to suggest on this question ?
Best,
References :
[1] Qu et al. 2012 (Experimental Hematology), Mesenchymal stem cells inhibit Th17 cell differentiation by IL-10 secretion
[2] Murray 2006 (Curr Opin Pharmacol) Understanding and exploiting the endogenous interleukin-10/STAT3-mediated anti-inflammatory response - see references 31-33
[3] Ng et al. 1997 (JBC) STAT3 Is a Serine Kinase Target in T Lymphocytes
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Hello Philippe,
The following 2 articles may be helpful to find the answer to your question:
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I've been attempting to quantify rat plasma cytokines following a mild infection. The low concentrations of the cytokines has made this very difficult using ELISA/LegendPlex techniques (I'm not detecting anything for the vast majority of my targets).
Does anyone know of a more sensitive technique to do this? I thought of using mass spec but this is expensive and I'd like to keep costs down if possible
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Did company suggested any positive controls, and did you use them, were you able to detect positive control
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Hi folks,
I extracted BAL fluid from mice to check total and differential leukocyte counts, as well as inflammatory cytokines and chemokines, and now I'm wondering how long I can keep my samples at -80 degrees to check the same. Suggest some practical experiences or methods for a better result.
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After collecting BALF, keep it on ice and do the cytospin within 2-3 h.
If you want to collect the total cells, after collecting BALF, centrifuge it at 5000 g for 10 min in a microfuge centrifuge. The pellet can be suspended back in ice cold physiological saline for staining and visualization under microscope. All these should be done keeping the samples on ice and within 2-3 h of BALF collection. I would suggest getting it done as son as BALF gets collected!
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I have been finding the proof that this epitope called NLVPMVATV does not induce cytokine storm for days, but in vain. Could anyone please help me? I really need a reference paper to support this or else my project will be in a great trouble.
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Hi,
Check the references below:
HCMV UL83/pp65 (NLVPMVATV)
CMV pp65 peptide NLVPMVATV (HLA-A*0201) is a single peptide for stimulation of T cells. The peptide from human cytomegalovirus (CMV) phosphoprotein (pp65) is synthesized as it is presented by the MHC class I HLA-A*0201 allele and can be used for targeted in vitro generation and expansion of antigen-specific CD8+ T cells.
Reference:
"Although the anti-pp65 antibodies and the pp65 antigens are detected in immune-depressed patients with active viral infection [16], the antibody response to the pp65 antigen in normal infected individuals is not always detectable by immunoblotting [17]. It has been reported that the pp65 antigen is targeted by a cell-mediated immune response and that its vaccination can induce a pp65-specific CTL response"
Reference:
Hope it helps,
Best regards
Tomasz
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I saw many papers are introducing or immunizing mice with ovalbumin (OVA), will this induce a cytokine storm? Is there any paper suggesting that OVA doesn't induce cytokine storm? I've been searching this for days, please help.
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No, there are essentially 3 major types of immune response to infection: an antiviral response which illicits interferon, a pro inflammatory response that makes il1, il6, tnfalpha etc wich is normally directed against bacterial and fungal infections ( and can lead to cytokine storm) and then the allergic response directed against allergens like ova that generates il4, il10, il13.
In mouse models, balb/c mice are typically used to study allergic responses as thy are inherently more allergic and c57bl/6 are used to study proinflammatory responses and diseases because they are inherently more prone to this type of immune reaction.
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There are many theoretical studies on the use of cytokines as an immunotherapy, how can this be applied in practice?
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Low-dose IL-2 for instance promote regulatory T cell function and may help to control chronic inflammatory diseases.
Given its anti inflammatory and suppressive functions IL-10 was considered to treat some chronic inflammatory conditions but clinical trials were not striking
These days the concept of immunotherapy has expanded and in the oncology field it is commonly associated with the use of immune chekpoint inhibitors which have been very succesful in some cancer types, particularly melanoma but many others. And more recently CAR-T therapies.
Theoretically, any manipulation of the immune system can be considered immunotherapy
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Context: You have two wells of PBMCs in a 200 uL culture medium. The cells in one well have been stimulated with an agonist (e.g. SEB) and the other has not. You want to assess cytokine production at 6, 12, and 24 hours. The cell numbers are extremely limiting and can't be plated into more wells.
In the above situation, is it okay to take, for example, 50 uL of the supernatant from each well at each time point (assuming an equal distribution of cytokine in the culture medium) and use these "aliquots" for cytokine analysis via ELISA? The medium would not be replenished at each time point.
Obviously, the total cytokine yield in the supernatant would be reduced (and would need to be corrected for later) but any changes in concentration would remain representative of the stimulation time. Is this rationale/thinking correct?
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Hey Nathan Kieswetter, you could use a Multiplex Platform like LUNARIS where you only need 3µl at each timepoint for up to 12 Cytokines. This won't disturb your PMBCs like loosing 50µl each time and you could get more insights from your precious cells.
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Hi all, I have several abcam cytokine ELISA kits that came with 2 lyophilized standard vials. I've already reconstituted one vial into stock concentration and I'm wondering if anyone has any experience with how long the stock is stable for. The website and product manuals don't seem to have an answer
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Upon reconstitution, the cytokine standard may be stored at 4°C between 2-7 days, and if it is for long-term future use it should be stored at -20°C. Please avoid repeated freeze-thaw cycles.
Best.
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Hi! I am new in flow cytometry and I would like to confront with someone who is more experienced than me.
I am currently carrying out a project to determine intracellular levels of several cytokines in PBMCs (both in monocytes and in lymphocytes), in two groups: patients vs healthy controls.
I will compare the percentage of cells able to produce certain cytokines (IFNγ-TNFα-IL-10-IL-6-IL1RA-IL1B-IL-17A) after being stimulated and, of course, stained.
1) My first question is: what controls is better to use in this protocol? I am thinking to use unstimulated and stained PBMCs to evaluate the ability of the immune system to respond after the stimulus compared to this basal situation. But apart from unstimulated samples, what other controls would you use (stimulated non stained as an indicator of autofluorescence? Isotype controls?)
2) If I use unstimulated samples to assess the basal situation, could I assume that the basal production of cytokines is zero? If not, what kind of negative control can I use to determine the basal production of a specific cytokine without the stimulus?
3) In patients, I could not assume that the basal production of cytokines is zero. In this case, is it correct to compare the basal production of patients’ cytokines with the basal production of healthy controls’ cytokines, even if the analyses will be conducted in different days?
Thank you very much for you attention and your precious help!
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1) Isotype controls are always the preferred controls for indicator of autofluorescence,confirming the proper channel compensations,and gating references,so I strongly recommend you to use ISO controls; Fluorescence minus one controls (FMO controls) can be an alternative;
2) You can't assume that the basal production of cytokines is zero, even of healthy samples. Actually, the problem is that FACS intracellular cytokines detection can not give you the answer like zero or not, it depende on how you perform and analyze, the answer is mostly relative. With a high possibility, Golgi inhibitors ( Golgi STOP or Golgi PLUG, etc) should be used for hours before staining for increasing the sensitivity of your FACS detection, in your experiments, the longer duration or relatively higher dosage of Golgi inhibitor you use, the relatively higher positive signals you will get, including your basal production group. So I recommend you to modulate the detection protocols ( especially the use of Golgi inhibitors) by preliminary experiments for the detection of basal production differences between healthy and patients.
3) So the stability of the used Flow Cytometer and consistency of your manipulation are quite important for this experiments. I think that the specific datas( MFI of cytokines etc) could be jumping, to a certain extent, but the differences between healthy and patients must be relatively stable. Paired comparision will help you to conduct the conclusion.
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I incubated the spleen cells isolated from C57BL mice in RPMI 1640 medium for 24 hours to acclimate to the medium. I then exposed my cells to LPS at different concentrations for 24 hours (0.1ug 1 ug and 2 ug). I also incubated my cells with LPS as a treatment at the same time as a live, CFS, and heat-killed bacterium. At the end of the incubation, I collected my supernatants and cleared my samples from cells and bacteria by centrifugation and stored my samples at -80 degrees.
However, there was no cytokine secretion in the ELISA assay (TNF-a and IFN-gamma)
I got positive results in cell viability and cell proliferation.
LPS increased cytokine release in RAW 264.7 cells specifically (TNF-a, IL-10 and IL1-B)
While my cells are alive and the LPS I use stimulates cytokine release in different cell lines, why doesn't it stimulate cytokine release in mouse spleen cells?
My mouse isolation protocol and references I use:
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Thank you so much it's can really help to me
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I want to expose my lung epithelial cells to different treatments in vitro and then detect pro- and anti-inflammatory cytokines/chemokines in the conditioned media - can I then do PCRs on the conditioned media? Any guidance would be very much appreciated!
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PCR is used to detect nucleic acids, not proteins. Generally, you would use either a Western blot or an ELISA to measure the amount of a secreted protein in cell culture media.
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Can we put the prepared medium containing stimulatory cytokines like MCSF or interleukins at +4C and re-use it again after few days or we should prepare new medium every time?
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Mohammad Aqdas Alright, That means I have to thaw and freeze the aliquoted cytokines more than 3 - 5 times because I use a small amount of them every time. I am afraid that the cytokines lose its activity when freezing and thawing 5 times.
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Is there a relationship between gene expression and the immune response to some pathological indicators in the human body, such as cytokines?
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If Im right in understanding the Q, DNA transcriptome can help you demonstrate each gene of interest ,including immune-related ones being expressed.
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Can anyone tell me how to stimulate pancreatic cancer cells for cytokine release in cell culture ?
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Hello,
I hope you are doing well. Please take a look at this link ( ).
Bests,
Pooya
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Hello, I am using the Multiplex ELISA Kit For Mouse Cytokine Panel 2 (4-Plex) purchased from Boster Bio to measure the four cytokines IL-6, IL-1B, TNF-a, and INF-y. In this plate, all four biomarkers for each cytokine are present in each well.
Boster Bio and its distributor Quansys Biosciences claim that I need an imager, a microplate reader that will actually capture an image of my plate, to be able to analyze the luminescence emitted by each cytokines' antigen.
I currently do not have access to an imager of this sort, and I am wondering if anyone has been able to circumvent this issue and was still able to use a normal microplate reader? Or, does anyone have any idea for how I should go about measuring each cytokines' luminescent response with a normal ELISA reader?
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Our team could not gain access to the manufacturer's imager, but we did use an imager by Cytation to get a sort of OD graph/picture that we used to validate our dilutions were in the standard curve range. Unfortunately, we did not get any quantification date. We are then using these dilutions in a different multiplex with a more compatible plate reader.
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Hello,
I did a cytokine quantification in human plasma with a custom procartaplex kit (thermofisher) on a Luminex xMAP (Bio-Rad). I have many samples that are below the detection limit (<OOR). How should I handle this data? Should I assign them to 0 value? to the detection limit ? exclude them? I have searched in several papers without really finding the answer to my question... would you know what would be appropriate in this situation?
Thank you and have a nice day,
Yann Dos Santos, PhD student
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Hello,
I have come across multiple articles where the <OOR could be replaced by the lower detection value for each of the cytokines assessed. This is usually the lowest standard concentration in some of the kits.
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Hello,
I have a question for you please.
to answer this scientific question: does the variation of cytokine levels (Il6) have an impact on the clinical score over time? we have the cytokine levels (quantitative variable), the clinical score (quantitative variable), and the measurements were done in 3 different times (v1, v2, v3)
Can you tell us which statistical test is adequate for this type of analysis?
Thank You
AHmed
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Perhaps you could use longitudinal path and/or cross-lagged panel analysis to address this question. Both are generalized (multivariate) regression types of analyses that allow you to analyze multiple independent and dependent variables as well as multiple time points in a single statistical model.
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In human PBMC cell cultures, is it necessary to use anti-Ig (in conjunction with other stimuli, such as IFNγ, IL-21and/or R848) to induce CD11c+T-bet+ B cells?? The cytokines and the TLR7-ligand alone , are not capable of inducing these cells??
Thank you in advance!!
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It really depends on the B cell population you begin the culture with. My cultures were sort purified prior to culturing. I found that BCR engagement was not absolutely necessary for cultures generated from memory subsets. Cultures that were initiated from naive B cells needed the BCR stimulation. However, the TLR7 and IL-21 signaling were essential for the development of the CD11c+ phenotype within the system.
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As we all know the complement is essential for innate/adaptive immune defense and also homeostasis and is upregulated during inflammation whether via classical (C4bC2a) or alternative (C3bBb) pathways. I was wondering if after inflammation (resolution) we have anti-inflammatory cytokines to counteract proinflammatory cytokines and efferocytosis ect to downplay inflammation, but what regulates or tones down complement-mediated inflammation?
Basically 1) Is there a time course on the levels of complement proteins (full length and cleaved) and 2) how are the levels regulated? (via liver or regional transcription/translation levels or degradation or inhibition?)
thank you in advance for any input and enlightenment as well as for pointing me to any references.
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Dysregulation of pro-inflammatory cytokines promotes immune-mediated injuries 1.
Both epithelial-cell proliferation and an increase in macrophages in the lung are associated with SARS 2. Pathogen-associated molecular patterns (PAMPs) such as 2019 novel coronavirus, proinflammatory cytokines, and lipopolysaccharide (LPS), cause macrophage transition. Macrophages activated in this transition termed M1 macrophages that promote inflammation 3. PAMPs are primarily sensed by members of the Toll-like receptor (TLR) family and this sensation activate transcriptional factor NF-κB that promotes secretion of proinflammatory cytokines 3. Activated or infected immune cells secrete excessive proinflammatory cytokines including MIP-1α, RANTES, IP-10, IL-1-6-8, TNF-α, TGF-β1, MCP-1and MCP-1 4. These cytokines promote severe lung injury 1.
Thalidomide is an immunomodulatory agent with strong antiangiogenic properties with COX-2 inhibitor celecoxib. Thalidomide inhibits the mRNA encoding such as TNF-α and VEGF. In addition, thalidomide modulates activated or irregulated NF-κB, resulting in suppressing severe lung injuries.
However, we are ordered not to conduct clinical trials with thalidomide and celecoxib
by Ministry of Health, Labor and Welfare, Japan. Because thalidomide is the dangerous drug.
I think it would be difficult to save the patients with severe pneumonia who could be saved with this regimen in the future. I would like you to recommend to the Japanese Ministry of Health, Labor and Welfare to take appropriate measures in Japan from the Form.
Please post your opinion in the form that Japanese people could have effective treatment for 2019-nCoV infection. https://www.kantei.go.jp/jp/forms/goiken_ssl.html
1. Jiang Gu , Christine Korteweg
Pathology and Pathogenesis of Severe Acute Respiratory Syndrome
Am J Pathol, 170 (4), 1136-47 Apr 2007
2. John M Nicholls , Leo L M Poon, Kam C Lee, et al
Lung Pathology of Fatal Severe Acute Respiratory Syndrome
Lancet, 361 (9371), 1773-8 2003 May 24
3. Yannic Nonnenmacher, Karsten Hiller
Biochemistry of Proinflammatory Macrophage Activation
Cell Mol Life Sci, 75 (12), 2093-2109 Jun 2018
4. Chaolin Huang, Yeming Wang,Prof Xingwang Li, et al
Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China
The Lancet Journal
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I'm trying to calculate the sample size I should have to study gene expression of IL-4 in asthmatic patients so I need a paper that is close to my work.
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You can use this study if you still do not calculate the sample size:
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I am planning on analyzing the cytokine expression of human bone marrow cells via Flow Cytometry. For conducting the intracellular cytokine staining, I am not sure what Protein Transport Inhibition and what kind of Cell Stimulation to use and in which concentration to dilute those. I am thankful vor any experience or input you might have.
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Hello Mats Urban ,
Q. What is the difference between the BD GolgiStop™ and BD GolgiPlug™ protein transport inhibitors?
A. The major difference between these two inhibitors is where they work within a cell. BD GolgiStop (monensin) works by accumulation of protein at the endoplasmic reticulum (ER) stage, while BD GolgiPlug (brefeldin) works with accumulation of protein at the golgi complex. The use of these inhibitors leads to an enhanced ability to detect cytokine-producing cells by intracellular flow cytometry.
Never worked with bone marrow cells before, only with PBMC. For intracellular cytokine staining, we used PMA/ Ionomycin/ Brefeldin-A (first paper) or PMA/ Ionomycin/ Monensin (second paper) and we are very happy with the results. PMA is a very potent cell activator (triggers maximal activation so you can see the "maximal" secretory potential of your cells).You can find the concentrations used by us in these articles.
About the concentrations, here's a snippet from the first paper:
Several concentrations of PMA/ION are report -ed in the literature for PBMC activation, rangingfrom 1 to 100ng/mL PMA (32-39) and from 0.4to 1.4μM for ION (that is roughly 300-1000ng/mL) (32-36,38), depending on protocol require-ments and personal preferences. While the exactPMA concentration values seem to be chosen ar-bitrarily (e.g. 1, 2.5, 5, 20, 25, 50 or 100ng/mL),most studies recommend that ION be added at aconcentration of 1μM, that is 710ng/mL. Also,the incubation time may vary between protocols,depending on the desired downstream applica -tion of the activated cells. Short-term activationrequires a minimum of 4-6h (32-38) and usual -ly employs higher concentrations of PMA/ION,while longer activation times may reach up to24-72h (33,37). BD makes recommendations on the optimal concentration of each ingredient forcellular activation purposes: PMA 5ng/mL, ion-omycin 500ng/mL and BD GolgiPlug 1μL/mLof cell culture.
About the type of protein transport inhibitor, there are several sources on-line saying that monensin is more toxic than brefeldin and also monensin does not completely block TNF secretion.
I hope this helps you at least to get an idea about this technique.
Good luck!
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I am wondering what happens to the frozen Conditioned-media (CM) that is collected from cancer cells and then incubated for 24 hours to 48 hours? Will the secreted proteins in CM such as Chemokines and cytokines be degraded? In other words, does the incubation affect the CM composition?
Many Thanks,
Fouzia
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In monolayer cell studies, CM is really nutrient depleted medium. This nutrient depletion causes the monolayer cells to spread out more. I.e., become flatter, which causes their nuclei to flatten and changes the chromosomes to change their distribution from 3D to 2D and hence, the number of their nearest neighbors. This halves the number of neighbors for chromosome exchanges and other interactions. The same effect can be produced by diluting fresh media with saline. See the my papers with N.M.S. Reddy, with Peter Mayer, with Joseph Ostashevsky, and with Maria Kapiszewska, all in the 1990s and early 2000s.
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I wanna selectively expand erythroblasts from freshly isolated PBMC for reprogramming into human iPSCs. Would I be able to substitute those above media with RPMI 1640 with cytokines and growth factors (SCF, IL-3, EPO, dexamethasone, IGF1 etc.) will be required for all media listed. Feel free to share your experiences or some literature references from your archive. Thanks in advance!
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Hi Evrim, Interesting work.
You know Marieke von Lindern from Sanquin from the Evidence meeting 2 years ago. https://www.researchgate.net/profile/Marieke-Lindern
I think this is something that she may be working on. So you might directly ask her with reference to you having met her for Evidence.
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As part of my research, I'm looking into the impact of this cytokine on HIF expression. When is the ideal time to evaluate HIF gene expression using qPCR and protein expression via Western blotting after IL-1B treatment of the A-549 cell line?
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Hello Ali. The treatment with IL-1B is based on the drug activity time. usually, to find the correlation between the HIF and IL-1B using qPCR and WB, you probably need to treat them at different time points. ( 1hr,4hr and 8hr) in low o2 conditions to ensure that the hypoxia state is maintained by the cells.
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Hi all,
I am currently looking into how the presence of an oncogene influences cytokines responsiveness (primes cells to be more responsive to cytokines) within RNA-seq data. What elements and tools should I be looking into?
Would upregulation transcription factors be something that would explain such a phenomenon? If so could someone direct me to a resource/literature that would possibly explain the relationship between transcription factors and cytokines responsiveness?
Thank you!
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Look at the properties of the STAT family and its canonical and non-canonical pathways.
good luck
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I want to test 8 antigens using in vivo (mice immunization and challenge) and in vitro assays (Elisa, elispot, growth inhibition, various cytokine conc measurements). Based on the data obtained and analyzed, 2 of these antigens will be selected for nonhuman primates (NHP) testing. My question is, How do I go about selecting the 2 antigens for the NHP experiments Do I use the elisa OD values, elidpot SI values, parasitaemia and cytokine conc values? If so, what will be the cut-off value for each assay? What if the different antigens perform differently in the different assays?
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I totally agree with colleaque Balam Saidou.Strain of causative agent of Malaria a parasite variant that is genetically unique and induce specific immune response expressed antigens in lab.animals(mice) that yield high immunogenicity are selectef as vaccine.mor detailed in attached ref.
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We observed increased levels of some anti Inflammatory cytokines in the serum samples of MDD patients. We hypothesised that neuroinflammation might involve in depression and in that case antiinflammatory cytokines were supposed to decrease during depression to support this hypothesis. Now how we can explain this elevated antiinflammatory cytokines in depression?
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Maybe, it is one of those games of the body. I mean, the working mechanism of the body generally does not fit in the mould. So if we ratiocinate in an orthodox way, we can miss some complicated mechanisms involved in the situation we consider. Therefore, with a shot in the dark, an increase of anti-inflammatory cytokines may stem from a compensation mechanism against the rise of inflammatory cytokines.
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Hello, I have a question while experimenting, so I have a question.
As a porous scaffold, fibroblasts were cultured using two products, Merck PS scaffold and Lenabio SeedEZ, and cytokine assay was performed with cell supernatant.
In the 2D culture system, there is no cytokine secretion even if the incubation time is long,
In the 3D culture system, the cytokine also increased according to the incubation time.
It seems to receive a lot of stress in 3D compared to 2D, but it is questionable whether it is basically this high even without irritating substances.
Even if it is not the corresponding scaffold, is there any researcher who has done a cytokine assay after incubation using this porous scaffold?
I'd love to hear what you think.
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Hi Yeaji,
I agree with Ankita that a cell-free control should be run to determine if the scaffolds are giving a positive signal with the assay. We have not had an issue with high baseline cytokine release in our internal testing using our SeedEZ scaffolds, although our work with fibroblast has been limited. I'd be happy to help you further if you provide some specifics about your experiment. Please reach out via our website and I will answer you personally.
Best wishes,
James
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Hello, I am planning an experiment to detect the immune response after cytokine stimulation. Does anyone inject cytokine i.v. into a mouse directly and be able to see effect after some time?
Thank you very much!
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Hello, everyone. recently I've been trying to establish passive EAE model by transfer Th17 cells polarized in vitro (from 2D2 mice). according to previous papers (like Ja¨ ger et al., 2009 ), 2d2 CD4 naive T cell should be isolated and then co-culture with APCs in the presense of Th17-differentiating cytokines and MOG35-55. Here what is the kind of APC? (many paper didn't mention). Would you like to choose CD11c+ cells?. By the way, sometimes it should be irradiated APC. what's the condition of irradiaiton and the cell types of APC?
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Really great work, congratulations, I am follow
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I will be collecting blood and separating plasma for a study. The location of the blood draw will be about 30 minutes away from the lab where the samples can be stored. I have the option of separating the plasma at the location of the blood draw and storing it in a freezer until it can be transported to the other lab, which has a -80 degree freezer. The caveat is that the freezer that would be used for temporary storage is a standard/regular freezer (not -80). Does anyone know if temporary storage in a regular freezer for a week or two prior to transferring to a -80 freezer would compromise sample integrity?
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Hi! In an ongoing project, we are also collecting blood to measure cytokines. usually I leave samples at 4.ºC up to 3 days before I separate the plasma and store them at -80.ºC, without any problem. I have already left samples for a couple days at -20.ºC before storing them at -80.ºC and we had no problems with the ELISA. For long-term storage -20.ºC is more "dangerous" but if we are talking about few days-1 week I think you will see no differences.
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We failed to stimulate THP-1 to produce cytokines limited poor cell membrane permeability of free cGAMP . Could you please give me some suggestions?
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Hi, try to make refreshing by culturing those cells in basic DMEM supplemented with human neonatal serum for three days. Later you can try to test it again.
Good luck.
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The production of cytokines is an important factor in the inflammatory process. On the other hand, natural products derived from medicinal plants appear to be (and often are) promising pharmacological tools to modulate cytokine production. Monorterpenes are promising substances that have modulated cytokine production in vivo (preclinical and clinical approach) (See: Monoterpenes modulating cytokines - A review. Food Chem Toxicol. 2019;123:233-257. doi: 10.1016/j.fct.2018.10.058 or Essential oils and its bioactive compounds modulating cytokines: A systematic review on anti-asthmatic and immunomodulatory properties. Phytomedicine. 2020; 73:152854. doi: 10.1016/j.phymed.2019.152854). The role of flavonoids is well known in the inflammatory process, especially when it comes to the production of cytokines (see: lavonoids as Cytokine Modulators: A Possible Therapy for Inflammation-Related Diseases. Int J Mol Sci. 2016;17(6):921. doi: 10.3390/ijms17060921 or Flavonoids as Th1/Th2 cytokines immunomodulators: A systematic review of studies on animal models. Phytomedicine. 2018;44:74-84. doi: 10.1016/j.phymed.2018.03.057). Several other natural products are promising, but how many of these have become drugs available on drugstore shelves? What is the biggest challenge to getting natural products that are modulators of important cytokines, such as cavacrol that modulates IL10 (Anti-inflammatory effects of carvacrol: evidence for a key role of interleukin-10. Eur J Pharmacol. 2013;699(1-3):112-7. doi: 10.1016/j.ejphar.2012.11.040), to be available for clinical use? Briefly share your opinion. I'm writing an article that thinks about this a bit and would like to read the opinion of other researchers who are interested in the topic.
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Who's the strongest?
what are the units that can express the role in immune reaction?
who can compare between them in a way that makes me able to the use
to be clear in what i teach
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doctor Gilles Marodon very thanks to you firstly..especially for simpling the sentence that is used.
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I'm design to culture mouse aorta in vitro and give some cytokines stimulation. After that, I will collect the vessle for parrfin section. I want to search some information for this, but there seems no articles on pubmed. So does anyone have some suggestions? Thanks so much.
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Is IL-37 anti-inflammatory or proinflammatory?
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IL-37 ( broadly suppresses innate inflammation and acquired immunity ) has the ability to inhibit the production of pro-inflammatory cytokines and ameliorate inflammation-induced fatigue by inducing metabolic reprogramming and limiting the metabolic effects of inflammation.
For more information, see www.hindawi.com
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I have been trying to check the expression of a pro-inflammatory cytokine between differentiated wild-type and knocked-down THP-1 macrophages cells. When I stimulate the cells (both at 24h and 6h)mRNA expression is reduced in kd-THP1 cells (by qPCR), but not the protein (checked by ELISA). The protein levels are similar between both the cells, when I have significant reduction in the mRNA levels. My question is, if anyone faced the same issue or any way that I can overcome this issue?
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This is not an issue at all and very common in secretome analysis. What matters for you is that both ELISA and western blot goes parallel with each other.
The explanation would be that your gene of interest is subjected to post transcriptional modifications, and that's normal and common (which is the case IL-6 for example)
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I am working on Adipogenesis. I interested in exploring inflammatory cytokines.
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Yes, may be
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I am trying to stimulate oral epithelial cells for cytokine release. I have tried different endotoxins, but not able to get any results.
Any suggestions?
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Hello Minati,
Do you use primary oral epithelial cells (OECs) from human donors, or do you use oral carcinoma-derived cell lines such as H413 or TR146?
The thing is, primary oral epithelial cells from human donors are naturally exposed to a variaty of different bacterial antigens, which are present in the oral mucosa and our food. These cells tolerate bacterial antigens, which are also present on commensal bacteria in our oral cavities, to avoid an over-reaction of the immune system. It will be difficult to get these cells to release cytokines.
I would suggest to use cell lines such as H413 or TR146 and different bacterial stimuli from both, gram+ and gram- bacteria.
In the following study, the authors used MV130, which is a cocktail of different heat-inactivated whole cell bacteria, to stimmulate H413, TR146 and also primary oral epithelial cells:
(Molero-Abraham et al. 2019) Human Oral Epithelial Cells Impair Bacteria-Mediated Maturation of Dendritic Cells and Render T Cells Unresponsive to Stimulation. Front Immunol. 2019 Jun 28;10:1434.
doi: 10.3389/fimmu.2019.01434
H413 and TR146 cells produced at least IL-6 and IL-8 after stimulation with MV130, whereas primary oral epithelial cells did not respond to MV130 stimulation.
I hope, this will help you.
Kind regards,
Alexander Gluschko
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Anyone have research done on how a cell responds differently if you inhibit the ligand (ie inhibit IL17 cytokine) versus inhibitor the receptor (ie. inhibit IL17RA).
If we inhibit a ligand, it cannot bind to the receptor to do it's function. If we inhibit the receptor the ligand cannot bind to do its function. Both may result in the same inhibition but in different mechanisms. I am looking into how these two mechanism can cause different results in disease (psoriasis).
If we inhibit the ligand, a cell can release more ligand. Likewise, if we inhibit the receptor, a cell can upregulate receptor expression to get more?
Thoughts?
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One important consideration is it there are may be multiple ligands for the same receptor, depending on the receptor of course. Blocking the receptor may therefore potentially inhibit the signalling from multiple ligands, rather than just one.
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Hello.
I try to detect the presence of cytokines IL-18 and IL-1beta by HEK-blue IL-18 and HEK-blue IL-1beta cells (cells develop by invivogene) these cells are transfected to detect the presence of IL-1b IL-18 in the supernatant. in order to maintain the stability of transfection, these cells are also transfected with resistance genes to selective antibiotics.
the question I ask is when should I add the selection antibiotic? do I have to add them 24 hours before the experiment or when the cells pass? if someone has worked with these cells or with other HEK cells give me a reply please.
thank you in advance.
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Dear Isilay, I recently noticed that Invivogen modified the HEK blue IL-1b cells. The "new" cells have the ordering no. #hkb-il1bv2 and therefore the new data sheet is valid: https://www.invivogen.com/sites/default/files/invivogen/products/files/hekblue_il1bv2_tds_0.pdf
For the "old" cell line with the ordering no. #hkb-il1b the medium I recommended below should be suitable. It might be the same with your cell line? You might contact the technical support for your issue. Sometimes it makes sence to reboot the cells for a few passages in medium without selection antibiotics till they are growing normally and then switch to selection medium afterwards.
I hope that helps!
Bests, Charlotte
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  • Epstein-Barr virus has proteins of sequence and functional homology with cytokines of the host. Due to cytokines mediating innate immune response, sustaining /or dysregulating of their actions result in hypercytokinemia which is known as cytokine storm syndrome. Cytokine storm is the symptom of hemophagocytic lymphohistiocytosis caused by Epstein-Barr viral infection where very high levels of cytokines are found in serum such as interferon-gamma which is more than 100 u/ml ( the normal is less than 1.0 u/ml), SCD 25 which is more than 10,000 u/ml ( the normal is less than 2000 u/ml ) as well as a significant rise in levels of IL -6, IL-10 and IL-18.
  • My question is, why cytokine storm syndrome which is known to be caused by the Epstein-Barr virus, now, have been correlated or linked to COVID-19?
References:
  1. -Sangueza Acosta M , Sandoval Romero E :Epstein -Barr virus and skin.Anais Brasileiros de Dermatologi. 2018;93(6) :786-799 .
  2. -Verbist K C ,Nichols K E : Cytokine storm syndromes associated with Epstein-Barr virus.Cytokine storm syndrome : Springer, 2019; 253 – 76.
  3. -Tangye S G , Palendira U, Edwards E S : Human immunity against Epstein -Barr virus -lessons from the clinic :Journal of Experimental Medicine,2017;214 (2): 269 – 83.
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Cytokine storm and cytokine release syndrome are life-threatening systemic inflammatory syndromes involving elevated levels of circulating cytokines and immune-cell hyperactivation that can be triggered by various therapies, pathogens, cancers, autoimmune conditions, and monogenic disorders. This is a very comprehensive definition taken form a review in the NEJM. I want to remark that Cytokine storm is defined in plural remarking that includes different mechanisms depending on the associated disorder or trigger. Currently everybody relates cytokine storm with Covid19 but cytokine storm is important in many other conditions including sepsis, CAR-T therapy, GVHD, and somehow macrophage activation ( haemophagocytic ) syndromes
I am attached some selected excellent, recent and comprehensive reviews
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Hello. I would like to assess the cytokines produced by cryopreserved PMBCs after PHA stimulation. More precisely, I want to measure intracellular cytokines using flow cytometry (with PMA/ION) (IFN-gamma, TNF-alfa, IL-17) and those from the cell medium using ELISA (IFN-gamma, TNF-alfa, IL-10). These are my queries
1.- I think that after thawing the PBMCs an overnight incubation without stimulus is a good idea; is that correct?
2.- Which PHA is better: PHA-P or PHA-L? Should I add IL-2 to the medium?
3.- Based on the literature, I think that 48 h stimulation is enough. However, when should I change the medium? Just the first day (the first overnight without stimulus)? And, what happens between the 2nd and 3rd day? Should I add more complete growth medium?
4.- Any other comment about this kind of experiment?
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1. Many people incubate cells after thawing. The "resting" period can be anywhere between 2h and 24h. I haven't found a paper about this topic yet, but from what i've read and my personal experience, 2h is the minimum and you should never let them rest for more than 24h because they start dying without stimulation. I would say anything between 2-24h should work. I think it's more important to choose a number and stick with it for all the samples in order to avoid an additional source of variability.
2. I've never worked with PHA so I have no opinion on this.
3. I don't know about the duration of stimulation, but I can say something about the cell medium. If you go for a 24h resting period, I think it would be best to change the medium before starting the stimulation assay. After that, if your cell medium is well prepared, you don't have to change it until the end. PBMCs at 1-2 million/mL can survive in proper culture media for more than 48h. There's enough nutrients for them. We use RPMI supplemented with 10% FBS and 1% anti-mycotic anti-bacterial solution. We frequently do cell cultures with 2 million PBMC/mL and they are just fine after 48h.
4. I think there are multiple ways of going at this. I am not sure how exactly you are willing to stimulate the cells with PMA, but I guess it's after the 48h of incubation with PHA?!
A short example of a protocol
- thaw PBMC
- 24h resting
- change culture medium
- add PHA
- 48h incubation with PHA
- change medium (and save the discarded one for ELISA if you want to do ELISA for the cytokines released during the 48h of incubation with PHA???)
- add PMA/ION (+Brefeldin A?) and incubate for 5h
- change the medium
- surface staining (if needed)
- cell fixation
- permeabilization
- intracellular cytokine staining
- data acquisition on a flow cytometer
This is a summary only. If you want to take a peek at our intracellular cytokine staining protocol after PMA/ION/BFA stimulation:
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Hi everyone,
I've been using proteome profiler kit from R&D to compare cytokine expression of tissue extracts from different treated and untreated mice.
Each replica is different from the previous one, I mean, it is not reproducible.
Has anyone used the #ARY006 kit, or other similar? Do you think it's useful?
Attached you can find two different replicas of my experiments.
Thanks
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I think you have a problem with the quality of the experiment. It looks like it has something to do with poor labeling of your samples. Do additional testing to make sure the protocol works in your hands. And ask the provider company R&D for help.
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I want to polarize THP-1 to M0, then to M2. After adding PMA and getting macrophages, I need to get the highest M2 percentage (90%) because I can not isolate M2 from other subtypes. For this reason, Does IL-4 alone enough to polarize macrophages to M2 or I need to use other cytokines like IL-13 or IL-10? in addition, what is the most practical CD marker for M2? CD206 or CD163? there are many researchers who say that it is not easy to get CD206 so should I use CD163 instead?
Thank you
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It depends on which M2 specific phenotype you are after. IL-4 and IL13 is enough to polarize macrophages to M2a. Similarly, stimulation with immune complexes lead towards M2b. Stimulation with IL10 and/or glucocorticoids leads towards M2c and with IL6 towards M2d activation !!
Both CD163 and CD206 are expressed by M2 macrophages but the expression of the receptors is differentially regulated. CD163 expression is increased in response to interleukin-10 (IL-10) stimulation, while CD206 expression is upregulated by IL-4 and IL-13. So, saying that it is difficult to see the expression of CD206 is not true.
BW
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I'm searching for a research article for cytokine study by ELISA for IL-6, TNF-a, IL-1B. Kindly recommend some good papers to proceed.
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I know rat antibodies are not well defined, but I will appreciate suggestions on which markers to look for IN RATS in case of the following:
1. activated T-cells
2. central memory T-cells
3. antigen-experienced T-cells
4. Th1, Th2, Th17 T-cell activation markers
Cell membrane or intracellular cytokine marker suggestions will be appreciated.
Thank you
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Please consider the following publications. In the methods section you have detailed lists of antibody cocktails and the respective clones used for flow cytometry.
I hope that helps.
All the best & good luck with your experiments,
Michael
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Dear coleagues,
Recently we acquired an ELISA kit for the detection of some cytokines with. It recommends reading the absorbance with the 405 nm wavelenght and apply correction with the 650 nm wavelength.
The one problem is that our plate reader does not have the 650 nm filter for reading, only 630 nm. As far as I've read, wavelengths of over 570 nm are often used for correction since they eliminate noise from the plate itself and other undesired components.
My question is would the difference be too significant between the correction wavelength of 630nm and 650? Could it interfere with our results? It's worth noting that our kit has HRP-linked avidin and recommends ATBS as the substrate.
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No, I would not expect any problems.
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Hi, dear γδ T cell researchers!
I'm looking for how to isolate Vdelta1 and Vdelta2 cells from peripheral blood and bone marrow, without stimulation, just isolate these two subtypes separately and then cultivate them and evaluate cytokine production.
I thought of isolating the total #gdTcell population first (using Anti-panTCRγδ magnetic beads) and then try to separate these two subtypes (Vd1 and Vd2) for culture in vitro, but I don't know how to proceed. I need to evaluate these subtypes without them being stimulated/activated.
I appreciate it if anyone can help me! ;)
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You can use fluorochrome-conjugated mAbs. First dye the sample with anti-CD45, anti-CD3, anti-Vd1 and anti-Vd2. I propose to use the Miltenyi, and use a MACSQuant® Tyto® cytometer. Second, you run the sample by positively selecting the Vd1 cells and obtain the Vd1 T lymphocytes in the positive well with sufficient purity, then you recover the sample from the negative fraction and run it again in another cartridge and positively select the Vd2 T cells. in this way you obtain the two populations of interest in a viable and quite simple way.
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Hi everyone,
Do you have any experience in preparing sample dilutions to measure the concentrations of inflammatory cytokines (TNFα, TGFβ, MCP-1, IL-1α, IL-1β, IL-6, and so forth) for an ELISA when it comes to any rat brain tissue, especially hippocampus. I'd prefer not to perform a pretest, and waste any well-strips beforehand. Based on your experience from different labs, what would be the expected protein concentration ranges of these cytokines in a supernatant saved from tissue homogenate prepared from a healthy rat (Sprague Dawley) brain tissue (hippocampus)?
Kind regards,
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Hello, I'm a student. I have a question that Why is IFN-gamma increasing while THP-1cell binding is reduced?
from the previous research Two very distinct cytokine secretion patterns have been defined amoung murine CD4+ T cells. Type 1 helper (TH1), but not type 2 helper (TH2), cells produce interleukin (IL)-2, gamma-interferon (IFN-γ) and tumour necrosis factor-β, whereas TH2, but not TH1, cells express IL-4, IL-5, IL-6 and IL-10. The different cytokine patterns lead to different functions of the two types of T cell. From the anti-inflammatory research I've read. He said Fucoxanthin could inhibit inflammation by reducing the binding of ThP1. cells, but with more IFN-gamma.
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Dear Chattramat!
Because IFN-γ reduces macrophage /monocyte motility.
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Hi everyone,
We'll perform an ELISA using rat hippocampi. As far as I know, it's recommended that a protease inhibitor cocktail be added to the lysis buffer prior to homogenization. When it comes to phosphatase inhibitors, I've heard that it's optional but not necessary. We think we'll evaluate some cytokines (TNF-alpha, IL-6, so forth) and some other proteins (PPAR-alpha).
So, would we still need to add protease inhibitors and phosphatase inhibitors to the lysis buffer together prior to homogenization for an ELISA? If not, what would be your rationale behind your opinion?
Best,
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Thank you for all your contributions, they are all appreciated. In relation with that, some scientists say some kits may not be compatible with protease inhibitor cocktails. How I could avoid making any mistake when deciding whether I use it or not? Are there any specific rules or limitations regarding protease inhibitor cocktails?
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Hi everyone,
Can someone share their optimal concentrations of PMA/ionomycin for spleen T cells?
I plan to use PMA/ionomycin as a positive control for T cell activation (surface markers, cytokines...)
Much help appreciated.
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We used PMA 50 ng/ml (Calbiochem, Billerica, MA) and 500 ng/ml ionomycin (Calbiochem) for C57Bl6/J derived T cells as per
There might be some value to do a titration experiment as the genetic background of the mice you are using, the environment and the specific functional readout (certain cytokines, co-stimulatory molecules, proliferation) may all be somewhat affected by the concentration you are using.
All the best & good luck with your experiment,
Michael
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Is there a possibility to treat cell first with serum, then wash (maybe with PBS) and after some hours evaluate cytokine/growth factors levels in supernatant (to exclude serum concentration) ?
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Hello Djúlio Zanin I remember reading something similar to this when I was trying to devise a PhD project on breast cancer. It has been done in breast cancer before where sera from patients was added to breast cancer cell-line. Also it has been done on metastatic cancers (DOI:10.1186/PREACCEPT-6395752331415020) and other cancers (PMID: 25750309). HBSS appears to be the wash of choice and then measurements can be performed. So, in theory your protocol should work well but you have to optimize exposure hours to determine the best window for assaying cytokine release, you don't want to get apoptosis cytokines due to prolonged HBSS (depends on the cell-line if sensitive to starvation) or second phase cytokines due to prolonged serum exposure and thus missing the first line induced release. Good luck
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It could be interested to analyse at the same pts some other variables such as cytokines (pro and antiinflammatory), apoptotic markers (AGEs, Fas Ligand and so on). Moreover if you have an MRI analysis you can test volumetric changes of different brain areas which are implicated to schizophrenic etiology.
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Same goes for BDNF and pro-inflammatory markers in MDD. Has anyone explored this relationship further?
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Hello guys,
I need some help with my WB troubleshooting.
I'm investigating Lung tissue of mice for different Cytokines.
In all my Westernblots from different samples I got strong non-specific bands , one between 150-100 kDa and on at 75 kDa and a faint one at 25kDA, which is not optimal because some of my Cytokines are in the same kDa range.
I have already done an WB only with the secondary antibody and apparently it's the cause of these non-specific bands, because I have got the same non-specific Bands like in my other WB. Even after I have changed to a different secondary Ab I have got the same results.
I did the block (1h 30min) with Milk 5% and I also tried BSA 5%.
All the first antibodies are incubated at 4 degree over night and the host is rabbit.
The secondary antibody (goat anti rabbit igG) was diluted in 5%Milk (TBS+ 0,1% Tween) 1:10000 and incubated for 1h.
Do you guys have any explanation for these non-specific bands.
The picture is from a WB which was only incubated with my secondary antibody. (I cut the Membran in to pieces)
Thank you guys for your help.
Kind regards
Marco
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Hello
You need to optimize the secondary antibody concentration. May be 1:10000 is too high. You can further dilute the secondary antibody, and you can go upto 1:20000.
Also, use affinity purified secondary antibody.
Good Luck.
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I am currently working on some ex vivo viral infection models with treatment with different agents. I found some pieces of information in the literature about pre-treatment with LPS to "strengthen" PBMC's (their viability) and cytokine release.
I usually seed cells on the plate, let them 16h of "rest", and then exposed them to the therapeutic agents (many configurations). I was wondering where to "put" that pretreatment with LPS and for how long should the cells be treated? I saw that some researchers add it before the treatment (which I assumed would be ok for my application), but some of them do it shortly after the treatment.
My idea was:
1. Thawing the PBMC's, adding LPS to the Falcone, leave for "rest day".
2. Wash the cells and seed them.
3. Treatment as usual.
What do you think about that? I have serious doubts if the cytokine profile will make sense after that combination.
Thank you in advance!
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Abdalsalam Kmail Thank you very much for your reply, I drew similar conclusions in the course of the further review, so your statement confirmed my assumptions.
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I'm working on two genes. one gene, along with other genes form cytokine gene cluster on the same chromosome. this gene particularly close to the other gene I'm working on also. This second gene, found to be regulated coordinately by several long-range regulatory elements in an over 120 kilobase range on the chromosome.
Can this affect its gene expression in SYBR green qPCR? knowing that I'm using the same cDNA template ?
It gives variated Cts upon replication.
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whether the regulatory elements are closed or away won't have influence on RT-qPCR since what you'll do is amplification only on cDNA. It sure will have a role on the transcription, but not on the measure of this expression.
if the CT are changing from sample to sample and replicates to replicates, maybe you should have a look somewhere else. see the quality of samples and replicates, all must be uniform. see also the quality of the primers design, maybe you could test them in in-silico PCR (as in UCSC or NCBI).
keep safe
fred
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Hi there. I wish to investigate macrophage function using intracellular flow cytometry for cytokines. 
Using our mouse model we harvest macrophages from various organs and analyse using standard flow. I want to look at their function using intracellular flow. It is hard to find protocols on this for macrophages. Protocols exist for t-cells but usually involve stimulating the t-cells then analysing in vitro. I am not sure if I would need to stimulate the macrophages after harvest from the mouse prior to flow, and what is the correct sequence for staining/permeabilisation etc.
If anyone has a protocol or can point me to a paper let me know thanks.
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Hi Nastaran,
I do not have the ready answer to your questions. At the same time, you could check the following topics discussing activation of macrophages:
In my experience, the rat resident peritoneal macrophages could die after stimulation with PMA exceeding conc. of 0.1 mg/L.
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I heard the best method in this case is euthanasia by decapitation. But should I use anesthetic agents? Doesn't it have an impact on the cytokines levels?
Thank you very much.
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We routinely use Isofluorane combined with decapitation for euthanisation of rats where blood or tissue samples are used for analysis. The process itself shouldn't last more than 60 seconds and has good reproducible results. Hope it helps.
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in the early onset of AID are you expected to be the anti-inflammatory cytokine to be increased or decreased?
if we find increased ( its correct to us to explain that as last resort of immune system to finish the fault immune response against self) but after the failure of this weapon, they start to decrease along with disease progression.
or can explain from another site of view ( high concentration of anti-inflammatory cytokines in the early onset of AID come from proteolytic cleavage to in situ high cytokine expression at the site of organ-specific AID and because of self cell destruction, then the cytokine leakage to peripheral blood and appear high in concentrations).
because as I read mostly AID researchers do not take into account the Chronology of autoimmune disease profile.
just take a snap for AID that anti-inflammatory cytokines decreased and proinflammatory increased...... that's logical but when that happens..?????
in my opinion, this picture reflects the last event when no more challenge from the immune system to correct the events.
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A lot of classical anti-inflammatory cytokines also have pro inflammatory effects ( example IL-10). so I would say it depends on which cytokines and what are the main players in AID You are looking at ?
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if there are membrane-bound and others soluble forms of an interleukin
as well as we find pro interleukin and mature form....
we know there are decoy receptors and ligands for cytokines
we know there are transiently elevated
we measure as researcher sometimes the interleukin in tissue as in immunohistochemistry
other estimated levels in biofluid like serum or saliva ......
my question starts here? are there any compensatory mechanism or hemostasis for cytokines?
(calcium and parathyroid hormones)
or difference between tissue expression and serum level ? or they (decrease/increased) in a parallel manner?
with best regard
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There is a great bias in the literature in understanding cytokines only as soluble factors in the blood, because that's what we can most easily measure. Few people even bother to assess what fraction of soluble cytokine is free and active, vs bound by soluble receptors and other binding proteins and inactive or modulated in some way.
That doesn't even scratch the surface of what's going on locally in the tissues. Often, local levels are high enough to match the receptor kD, but serum levels are not. You could view the serum levels as waste cytokine that's on the way out, after it did its job in a locally intelligent manner in the tissues. We measure them when the job is done, and their status no longer particularly matters.
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I made an cytokines expression curve, in 7dpi and 14 dpi Interferon gamma have a expression peaks, but at 28 dpi I observed an expression below the mock / control. Could this result be only related to the resolution of the viral infection, or a possible way of evading the immune response?
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Just assuming- It may happen due to immune cell exhaustion. The producers of IFNg like Helper T cells, NK cells may be in a dampened functional state after a massive host-pathogen battle.
Does the virus directly affect these IFNg producing cells? Then the virus can remain in a latent state and downregulate immune function.
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what criteria used to select the best standard curve fitting in ELIZA
logistic
cubic
cubic spline
linear?
sunlong ELIZA cytokine analysis
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Dear Kaled,
I confirm and agree with answer of doctor Waleed Alnasrawy.
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Hello,
I am new to calcium imaging data analysis, and I need to analyse it for my master's project (I didn't actually acquire the calcium images myself). So in the experiments, the primary mouse neurons were pre-treated with one of the three possible treatments (vehicle, cytokine alone, and cytokine + its receptor inhibitor) 24 hours prior to the imaging. Then, on the next day, the neurons were loaded with Fluo-4 calcium indicator and imaged for 5 mins. KCl treatment was also used as a positive control, but unlike with other treatments, neurons were not pre-treated with KCl for 24 hours. Instead, KCl was added to the neurons 1 min after the imaging was started. I used the software for the microscope to obtain the time measurements of changes in Fluo-4 intensity separately in somas and neurites.
So for KCl, I was told to use the mean of the intensity before KCl addition (i.e. 1 min) as F0 and divide all the intensities by the corresponding F0 values (i.e. F/F0), but I am not sure what to do with the other treatments since there isn't a clear period which I can use as an F0 to normalise them. I am also not sure if I need to normalise these at all. I would also appreciate any suggestions on the type of statistical analysis that I should use to compare these treatments.
Thank you in advance for your help!
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Thank you very much for your response! So just to clarify, I was only able to calculate F0 for cells that were acutely treated with KCl (which is our positive control) 1 min after the imaging was started. So I used the mean intensity before 1 min as F0 for these KCl-treated cells only. For the rest of the conditions (cytokine, cytokine+inhibitor, and vehicle), the cells were pre-treated with these agents 24 hours before the imaging, so there wasn't any acute addition of any agents during the imaging. I also do not have any completely untreated cells to use as F0, as you've suggested, so I am not sure what would I use as F0 (if anything) for my 24 hrs pre-treated cells? But your suggestion on using the vehicle for normalisation of cytokine treatment makes a lot of sense and I will discuss it with my supervisor, so thank you so much for it, it is very helpful!
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I performed the multiplex cytokine bead array (Thermofisher Procartaplex 20 plex) for plasma samples. However, I forgot to mix the contents while loading the standard 1 in the first well (A1) of the 96-well plate. Due to this, the standard curve formed is a bit deviated because of the standard concentration value of A1. I have attached the image of the standard curve formed. Please suggest how do I analyze the data.
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@Wolfgang Schechinger Ok Sir. I'll try.
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Working with murine spleenocytes. I stimulate 1 million cells with 50ng/ml PMA, 2ug/ml Ionomycin, 10ug/ml Briefeldin A for 4 hours at 37degC 5%CO2, followed by intracellular staining for cytokines, primarily IFNy. Unstimulated cells show more production of cytokines than stimulated cells. Is it better to add BFA along with PMA/Ionomycin or should I incubate cells with PMA/Ionomycin for 1-2 hours before adding BFA? When exactly should BFA be added?
Any other tips?
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Hi I have tried staining IFNg both overnight (+16h) and for 4h. Overnight stimulating woth IL12/18 showed increased IFNg expression compared to 4h. So I think you can keep the 4h of BrefA or Monensin incubation, but the stimulation time should be longer.
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I have been using THP1 cell lines. We use PMA (24 h treatment) for the differentiation of Monocytes into macrophages. But in my experiments, when I stimulate the macrophages with cytokines, i do not get the required results. The results are aberrant, like the changes in cytokine production are not in an order. After 24 hr PMA treatment, in our lab, we do not change the media (having PMA) and directly add the specific cytokine for investigating their effects.
Here, i want to know if the PMA is media is interefering with the stimultatory effect of our cytokine? I couldn't find any literature. If there is any literature evidence, please share.
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Thank you so much @Maria
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I am working on cell culture supernatant. Is that Ok if I store supernatants at -20C instead of -80 for cytokines measurement with ELISA assay? .
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Dear Maryam,
I agree with Marc Herb. If you want to store cell culture supernatants for 6 months, it would be better to store in -80°C. Furthermore, it is better to aliquot the supernatants to avoid multiple freeze-thaw cycles.
Best wishes,
NH
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Immune response against viral infection is cell mediated which releases many cytokines. It is clear that, in covid-19 infection level of cytokines is very high and called ' cytokine storm". Complications due to covid-19 infection is mostly due to this cytokines and can endanger the life by internal blood clotting. Other than remission of fever paracetamol has no anti-inflammatory action and this drug has no role on preventing release of cytokines. So use of paracetamol in this case will decrease fever escaping cytokines complications. On the other hand, along with action of decreasing fever NSAID can help to block formation of cytokines and thus prevents much of the complications due to covid-19 infection and decrease mortality. No doubt steroid is better than NSAID in this respect but many physician will hesitate to use steroid initially without confirmation. In all cases famotidine should replace PPI.
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Not at all . Paracetamol is safer than NSAID ( severe side effects and not to be used for ulcer , kidney pateints ) . HChQ is better than NSAID to reduce inflammation of Covid19 .
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My unstimulated PBMCs (thawed) show a better differentiation and cytokine profile on facs than my E.coli (heat inactivated) stimulated ones (but less than my CD3/28 stimulated positive controls). I have changed media, used different reagents, hoods, autoclaved everything I can think off but not sure why this could be. anyone experienced anything similar or had any ideas for why this might be happening please?
thanks
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the only other issue is that I cultured the same cells with inactivated E.coli and I observed less of a response - why would this be if the whole batch was contaminated? should the PBMCs not respond equivocally?
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I am determining cytokines TNF-a, IL-6, IL-1b, IL-10 y IL-15 in serum using Bio-Plex Pro human cytokine Screening panel Kit of Bio-Rad. However, I have had troubles with IL-15 determination. I had added a standard curve point to detect the low concentration of this cytokine. Also, I tried several dilutions of serum (sample without dilution, 1:2, 1:3, and 1:4) without success.